Treatment of tumors with miRNA targeting CDK4/CDK6

ABSTRACT

The present disclosure provides miRNA mimics targeting CDK4 and/or CDK6, and compositions comprising the same. Methods for the treatment of tumors, including but not limited to colon cancer, comprising administering a modified oligonucleotide comprising a miRNA are also disclosed herein.

FIELD

The present disclosure is directed, in part, to compositions and methodsand for the treatment of tumors and cancer, wherein the methods compriseadministering a modified oligonucleotide comprising a miRNA targetingCDK4 and/or CDK6.

BACKGROUND

miRNAs are 18-22 nucleotide small non-coding RNAs that can inhibittranslation and/or affect mRNA stability by binding to the 3′untranslated region (UTR) of their target genes (Ameres et al., Nat.Rev. Mol. Cell Biol., 2013, 14, 475-88; and Friedman et al., GenomeRes., 2009, 19, 92-105). Studies in different tumor models have shownthat miRNAs can either be oncogenic, as in the case of miR-155, miR-21and miR17-92 cluster, or tumor suppressors, such as miR-34a, let-7family and miR-143 (Iorio et al., Cancer J., 2012, 18, 215-22; and Oomet al., Biomed. Res. Int., 2014, 2014, 959461).

There were an estimated 134,490 cases of colorectal cancer (CRC)diagnosed in the U.S. in 2016 and an estimated 49,190 deaths accordingto the National Cancer Institute (NCI) Surveillance, Epidemiology, andEnd Results (SEER) program, and the 5-year survival for advanced (stageIV) disease remains low at 13.5% (Howlader et al. (eds), SEER CancerStatistics Review, National Cancer Institute, Bethesda, Md., 1975-2016).New treatments are needed to address the mortality and morbidity fromadvanced CRC.

The p16-CDK4/6-Rb pathway is an important target in oncology as it is acentral regulator of the mammalian cell cycle in the G1-phase (Malumbreset al., Nat. Rev. Cancer, 2001, 1, 222-31; Ruas et al., Biochim Biophys.Acta, 1998, 1378, F115-77; and Xiao et al., Mol. Biol. Cell, 2011, 22,3055-69). CDK4 and 6 are important targets in cancer and small moleculeinhibitors have been development and some have been approved by the Foodand Drug Administration (FDA) as cancer therapeutics (Peyressatre etal., Cancers (Basel), 2015, 7, 179-237; Shapiro, J. Clin. Oncol., 2006,24, 1770-83; and Sherr et al., Cancer Discov., 2016, 6, 353-67).

Dysregulation of genes in the CDK4/6-Rb cell cycle pathway is anindicator of cancer (Hanahan et al., Cell, 2011, 144, 646-74). Cancercells subvert different cell cycle checkpoints to continue uncheckedgrowth and proliferation. These mechanisms of overcoming cell cyclecheckpoints are mutually exclusive and can involve loss oftumor-suppressor proteins such as p16 INK4A or Retinoblastoma (Rb) oramplification of oncogenes like CDK4, CDK6, Cyclin D. Compared to lossof Rb, inactivation of p16INK4A by homologous deletion, frameshiftmutations or methylation are more common events (Ruas et al., BiochimBiophys. Acta, 1998, 1378, F115-77; and Jiang et al., Mol. Cell. Biol.,1998, 18, 5284-90). Loss of p16INK4A permits escape from senescence andprovides an advantage for tumor progression. Further, this leaves cancercells with increased levels of CDK4, CDK6 and Cyclin D-dependent kinaseactivities. Tumors like melanoma and CRC, however, show amplification ofCDK4 and/or CDK4R24C mutations (loss of INK4 binding site). Hence,ongoing clinical trials for CRC patients with advanced disease areevaluating the efficacy small molecule inhibitors of CDK4/6 as singleagents and in combination with chemotherapy. Emergent knowledge withCDK4/6 inhibitors in clinical trials indicates that these therapies havelimitations as monotherapy (Garber, Science, 2014, 345, 865-7). Thus,there is certainly need to develop alternate strategies to target CDK4/6and identify biomarkers of response, which is critical for understandingthe clinical results seen with CDK inhibitors.

miRNA mimics are emerging therapeutics that can target multipleoncogenes and thus have broad mechanisms of anti-cancer effects.Further, they can serve as biomarkers of response and help predictresponse.

SUMMARY

In the present disclosure, novel miRNAs that target CDK4/6 and which maybe used for therapeutic purposes have been identified. A family of fourmiRNAs that target CDK4 and CDK6 and lead to anti-proliferative effectsin CRC cell lines is described herein. In addition, synergies with, forexample, irinotecan and 5-fluorouracil, drugs used to treat CRC in theclinic, have been observed.

The present disclosure provides modified oligonucleotides consisting of15 to 40 linked nucleobases, or a salt thereof, wherein theoligonucleotide comprises a nucleobase sequence that is at least 80%identical to a nucleobase sequence of hsa-miR-6883-5p, hsa-miR-149-3p,hsa-miR-6785-5p, or hsa-miR-4728-5p.

The present disclosure also provides pharmaceutical compositionscomprising any one or more of the modified oligonucleotides describedherein which consist of 15 to 40 linked nucleosides, or a salt thereof,wherein the oligonucleotide comprises a nucleobase sequence that is atleast 80% identical to a nucleobase sequence of hsa-miR-6883-5p,hsa-miR-149-3p, hsa-miR-6785-5p, or hsa-miR-4728-5p.

The present disclosure also provides methods for treating tumors orcancer comprising administering to a subject in need thereof any one ormore of the oligonucleotides described herein, which consist of 15 to 40linked nucleobases, or a salt thereof, and/or a pharmaceutical agentthat induces the production of the one or more oligonucleotides and/orinduces PER1 expression, wherein the oligonucleotide comprises anucleobase sequence that is at least 80% identical to a nucleobasesequence of hsa-miR-6883-5p, hsa-miR-149-3p, hsa-miR-6785-5p, orhsa-miR-4728-5p.

The present disclosure also provides methods for treating tumors orcancer comprising administering to a subject in need thereof apharmaceutical composition comprising any one or more of theoligonucleotides described herein, which consist of 15 to 40 linkednucleobases, or a salt thereof, and/or a pharmaceutical agent thatinduces the production of the one or more oligonucleotides and/orinduces PER1 expression, wherein the oligonucleotide comprises anucleobase sequence that is at least 80% identical to a nucleobasesequence of hsa-miR-6883-5p, hsa-miR-149-3p, hsa-miR-6785-5p, orhsa-miR-4728-5p.

These and other embodiments of the present disclosure will becomeapparent in conjunction with the figures, description and claims thatfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, and 1D show: RNA expression data from TCGA CRC patientsamples showing expression of CDK4 and CDK6 in 50 tumor and normalsamples (FIG. 1A); TargetScan analysis of putative binding site(s) offamily of four miRNAs in the 3′UTR regions of CDK4 and CDK6,respectively (FIG. 1B); scatter plot of expression of miR-149* in 11 CRCpatient tumors compared to matched normal tissue (FIG. 1C, left panel);histogram of CDK4/6, p16 and Rb status in the same 11 patients (FIG. 1C,right panel); and a box plot of the log₁₀ RNA expression of PER1 genesin the same 50 normal samples compared to tumor samples (FIG. 1D).

FIGS. 2A, 2B, and 2C show: CDK4 and CDK6 protein levels in a panel ofCRC cell lines reverse transfected with 50 nM of miRNA mimics orscrambled duplex (SCR) for 72 hours (FIG. 2A); qRT-PCR for CDK4 and CDK6in SCR or miRNA mimics transfected in CRC cell lines (FIG. 2B); andmeasured luciferase activities of HCT-116 cell stably selected withCDK4-Luciferase construct reverse transfected with either SCR or 50 nMof indicated miRNA mimics for 48 hours (FIG. 2C).

FIGS. 3A, 3B, 3C, 3D, and 3E show: effects on cell viability measured 72hours post-transfection of CRC cell lines reverse transfected witheither 50 nM SCR or 50 nM of indicated miRNA mimics using CellTiter-Gloassay Z (FIG. 3A); effects of miRNA mimics on long-term cellproliferation of CRC cell lines reverse transfected with 100 nM of SCRor indicated miRNA mimic assessed by colony formation assays;representative images of cells stained with crystal violet are shown(left panel) and relative colony number is represented graphically(right panel) (FIG. 3B); representative western blots showing effects oncell cycle markers (FIG. 3C) and markers of apoptosis (FIG. 3D); andrepresentative results of changes in G1 and sub-G1 phases of cell cycleand apoptotic cells assessed in three CRC cell lines reverse transfectedwith either SCR or 50 nM (HCT-116 and HT-29) or 100 nM (RKO) miRNAmimics (FIG. 3E).

FIGS. 4A, 4B, 4C, and 4D show: effects of short-term cell proliferationof four CRC cell lines reverse transfected with SCR or 80 nM of CDK4 orCDK6 siRNA measured 72 hours post-transfection using CellTiter-Glo assay(FIG. 4A); representative images of cells reverse transfected with 80 nMCDK4 or CDK6 and stained with crystal violet (FIG. 4B); representativewestern blots of four CRC cell lines reverse transfected with 80 nM CDK4or CDK6 siRNA (FIG. 4C); and representative results of changes G1 andsub-G1 phases of cell cycle and apoptotic cells assessed in three CRCcell lines by reverse transfecting with SCR or 40 nM (HCT-116) or 80 nM(HT-29 and RKO) siRNA of CDK4 and CDK6 (FIG. 4D).

FIGS. 5A, 5B, 5C, and 5D show: cell viability 72 hours post-transfectionof a panel of CRC cell lines reverse transfected with 25 nM (HCT-116) or50 nM of SCR or indicated miRNA mimics, with addition of the indicateddoses of Irinotecan (FIG. 5A) or 5-FU (Figure B), using theCellTiter-Glo assay; and representative western blots of cells treatedwith 50 nM of miRNA mimic and Irinotecan (FIG. 5C) or 5-FU (FIG. 5D)showing the effect on apoptosis and cell cycle markers.

FIG. 6 shows a Western blot of the expression of p16 in CRC cell lines,and pancreatic cancer cell lines HPAF-II and BxPC3 as positive controls.

FIG. 7 shows sequence similarity of miRNA family assessed by sequencealignment using CLUSTAL-W (hsa-miR-6883-5p is SEQ ID NO: 1;hsa-miR-149-3p is SEQ ID NO: 2; hsa-miR-6785-5p is SEQ ID NO: 3; andhsa-miR-4728-5p is SEQ ID NO: 4); * marks indicate nucleotide identity.

FIG. 8 shows RNA expression data from TCGA CRC patient samples showingexpression of CDK4, CDK6 and PER1 in matched 50 tumor and normalsamples; scatter plots indicate the log₁₀ RNA expression of normalsamples compared to tumor samples for the indicated gene of interest;p-values were obtained from the Wilcoxon test for paired samples and areindicated.

FIG. 9 shows cell viability, cell proliferation, and western blotanalysis of pancreatic cancer cell lines treated with SCR or theindicated miRNA.

FIG. 10 shows cell viability and western blot analysis of melanomacancer cell lines treated with SCR or the indicated miRNA.

FIG. 11 shows cell viability and western blot analysis of the inductionof G1-cell cycle arrest by targeting CDK4 and CDK6, independent of p53status.

DESCRIPTION OF EMBODIMENTS

In the present disclosure, computational and TCGA analyses was performedto identify novel miRNAs that can target CDK4/6 and that can be used fortherapeutic treatment of colorectal cancer (CRC). The 3′-UTR of CDK4/6mRNA is shown here to be novel targets of a previously uncharacterizedfamily of miRNAs encompassing miR-6883-5p, miR-149*, miR-6785-5p, andmiR-4728-5p. The data presented herein of miRs 6883-5p and 149* revealedthat both miRNAs downregulate CDK4 and CDK6 protein and mRNA expressionwhen ectopically expressed in human CRC cell lines. RNA-seq dataindicated an inverse relationship between the expression of CDK4/6 andmiR-149* and intronic miRNA-6883-5p encoding gene PER1 in CRC patientsamples. Restoring expression of miRs 6883-5p and 149* had significantanti-proliferative effects, G0/G1-arrest, and apoptosis in CRC celllines. Targeting of CDK4/6 by miR-6883-5p and miR-149* can only, inpart, explain the anti-proliferative effects of these miRNAs as seen onsilencing CDK4/6 in CRC cell lines. Lastly, both miRNAs synergized withfrontline CRC chemotherapy irinotecan and sensitized mutant p53 celllines to 5-FU. Thus, the miRNA-based therapeutic strategy to targetCDK4/6 can be used as both a single agent and combinatorial therapy, andto identify biomarkers of response.

In particular, in the present disclosure, a combination of in silicoprediction algorithms and TCGA analysis was used to identify tumorsuppressor miRNAs that can translate to single agent and/orcombinatorial therapies for CRC. There have been previous studiesdescribing miRNAs regulating of CDK4 expression in melanoma and NSCLC(Georgantas et al., Pigment Cell Melanoma Res., 2014, 27, 275-86; Lin etal., Cancer Res., 2010, 70, 9473-82; Shao et al., Oncotarget, 2016, 7,34011-21; and Deng et al., J. Neurooncol., 2013, 114, 263-74). However,there are no such tumor suppressor miRs identified in CRC and/or studiesevaluating their use as therapeutics. In the present disclosure, a newfamily of miRNAs, whose expression is lost in CRC, has be identified.The primary gene network (i.e., G1-S phase of cell cycle, which isregulated by two of the four miRNAs in the family) was characterizedherein. Within the CDK4/6-Rb pathway, the present disclosure presentsdata that gene targets mediating the anti-proliferative effects ofmiR-6883-5p and miR-149* include CDK4, CDK6 and FOXM1, which hasrecently been identified to be phosphorylated by CDK4/6 (Anders et al.,Cancer Cell, 2011, 20, 620-34). FOXM1 is a Forkhead Box familytranscription factor that has also been also linked to resistance todifferent chemotherapies like cisplatin and 5-FU (Wang et al., LungCancer, 2013, 79, 173-9) and is an important oncogenic target inpreclinical development. Thus, the inhibition of CDK4/6 and itsdownstream effector FOXM1 by miRNAs has an advantage of targeting theCDK4/6-Rb pathway at different levels. The combination experiments withFDA-approved chemotherapies Irinotecan and 5-FU, as demonstrated herein,show that both the miRNAs can sensitize CRC cell lines to each of thedrugs and indicate that the use of combinations of miRNAs as adjuvanttherapeutics for the treatment of colorectal cancer is a viable clinicalstrategy. In addition to cell cycle, the pro-apoptotic functions ofmiR-6883-5p and miR-149* has been demonstrated herein. Both miRNAs, bydownregulating anti-apoptotic proteins BCLxL and XIAP, lead to apoptosisboth as single and combination in CRC cell lines.

In summary, new miRNAs, including miR-6883-5p and miR-149*, have beenidentified herein as direct negative regulators of CDK4 and CDK6.Restoring the expression of miR-6883-5p and miR-149* in cancer cellsshowed anti-proliferative effects and apoptosis as single agents and incombination, respectively. Thus, miRNA mimics as adjuvant therapy forcancer is an alternate to small molecule inhibitors.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in thearts to which the claimed subject matter.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

As used herein, “colon cancer” means malignancy of the colon, either aprimary cancer or metastasized cancer.

As used herein, “subject” means a human or non-human animal selected fortreatment or therapy.

As used herein, “in need thereof” means a subject identified as in needof a therapy or treatment. In some embodiments, a subject has a tumor orcancer, such as colon cancer. In such embodiments, a subject has one ormore clinical indications of a tumor or cancer, such as colon cancer, oris at risk for developing a tumor or cancer, such as colon cancer.

As used herein, “administering” means providing a pharmaceutical agentor composition to a subject, and includes, but is not limited to,administering by a medical professional and self-administering.

As used herein, “parenteral administration,” means administrationthrough injection or infusion. Parenteral administration includes, butis not limited to, subcutaneous administration, intravenousadministration, or intramuscular administration.

As used herein, “subcutaneous administration” means administration justbelow the skin.

As used herein, “intravenous administration” means administration into avein.

As used herein, “intratumoral administration” means administrationwithin a tumor.

As used herein, “intraperitoneal administration” means administrationinto the peritoneum (i.e., body cavity).

As used herein, “chemoembolization” means a procedure in which the bloodsupply to a tumor is blocked surgically, mechanically, or chemically andchemotherapeutic agents are administered directly into the tumor.

As used herein, “duration” means the period of time during which anactivity or event continues. In some embodiments, the duration oftreatment is the period of time during which one or more doses of apharmaceutical agent or pharmaceutical composition are administered.

As used herein, “therapy” means a disease treatment method. In someembodiments, therapy includes, but is not limited to, chemotherapy,surgical resection, and/or chemoembolization.

As used herein, “treatment” means the application of one or morespecific procedures used for the cure or amelioration of a disease. Insome embodiments, the specific procedure is the administration of one ormore pharmaceutical agents.

As used herein, “amelioration” means a lessening of severity of at leastone indicator of a condition or disease. In some embodiments,amelioration includes a delay or slowing in the progression of one ormore indicators of a condition or disease. The severity of indicatorsmay be determined by subjective or objective measures which are known tothose skilled in the art.

As used herein, “prevention” refers to delaying or forestalling theonset or development or progression of a condition or disease for aperiod of time, including weeks, months, or years.

As used herein, “therapeutic agent” means a pharmaceutical agent usedfor the cure, amelioration or prevention of a disease.

As used herein, “chemotherapeutic agent” means a pharmaceutical agentused to treat cancer.

As used herein, “chemotherapy” means treatment of a subject with one ormore pharmaceutical agents that kills cancer cells and/or slows thegrowth of cancer cells.

As used herein, “dose” means a specific quantity of a pharmaceuticalagent provided in a single administration. A dose may be administered intwo or more boluses, tablets, or injections. In some embodiments, a dosemay be administered in two or more injections to minimize injection sitereaction in an individual.

As used herein, “dosage unit” means a form in which a pharmaceuticalagent is provided. In some embodiments, a dosage unit is a vialcontaining lyophilized oligonucleotide. In some embodiments, a dosageunit is a vial containing reconstituted oligonucleotide.

As used herein, “therapeutically effective amount” refers to an amountof a pharmaceutical agent that provides a therapeutic benefit to ananimal.

As used herein, “pharmaceutical composition” means a mixture ofsubstances suitable for administering to a subject that includes apharmaceutical agent. For example, a pharmaceutical composition maycomprise a modified oligonucleotide and a sterile aqueous solution.

As used herein, “pharmaceutical agent” means a substance that provides atherapeutic effect when administered to a subject.

As used herein, “active pharmaceutical ingredient” means the substancein a pharmaceutical composition that provides a desired effect.

As used herein, “metastasis” means the process by which cancer spreadsfrom the place at which it first arose as a primary tumor to otherlocations in the body. The metastatic progression of a primary tumorreflects multiple stages, including dissociation from neighboringprimary tumor cells, survival in the circulation, and growth in asecondary location.

As used herein, “overall survival time” means the time period for whicha subject survives after diagnosis of or treatment for a disease.

As used herein, “progression-free survival” means the time period forwhich a subject having a disease survives, without the disease gettingworse. In some embodiments, progression-free survival is assessed bystaging or scoring the disease. In some embodiments, progression-freesurvival of a subject having colon cancer is assessed by evaluatingtumor size, tumor number, and/or metastasis.

As used herein, “improved colon function” means the change in colonfunction toward normal limits.

As used herein, “acceptable safety profile” means a pattern of sideeffects that is within clinically acceptable limits.

As used herein, “side effect” means a physiological responseattributable to a treatment other than desired effects. In someembodiments, side effects include, without limitation, injection sitereactions, colon function test abnormalities, renal functionabnormalities, liver toxicity, renal toxicity, central nervous systemabnormalities, and myopathies. Such side effects may be detecteddirectly or indirectly.

As used herein, “injection site reaction” means inflammation or abnormalredness of skin at a site of injection in an individual.

As used herein, “subject compliance” means adherence to a recommended orprescribed therapy by a subject.

As used herein, “comply” means the adherence with a recommended therapyby a subject.

As used herein, “recommended therapy” means a treatment recommended by amedical professional for the treatment, amelioration, or prevention of adisease.

As used herein, “targeting” means the process of design and selection ofnucleobase sequence that will hybridize to a target nucleic acid andinduce a desired effect.

As used herein, “targeted to” means having a nucleobase sequence thatwill allow hybridization to a target nucleic acid to induce a desiredeffect. In some embodiments, a desired effect is reduction of a targetnucleic acid.

As used herein, “modulation” means to a perturbation of function oractivity. In some embodiments, modulation means an increase in geneexpression. In some embodiments, modulation means a decrease in geneexpression.

As used herein, “expression” means any functions and steps by which agene's coded information is converted into structures present andoperating in a cell.

As used herein, “nucleobase sequence” means the order of contiguousnucleobases, in a 5′ to 3′ orientation, independent of any sugar,linkage, and/or nucleobase modification.

As used herein, “contiguous nucleobases” means nucleobases immediatelyadjacent to each other in a nucleic acid.

As used herein, “percent identity” means the number of nucleobases infirst nucleic acid that are identical to nucleobases at correspondingpositions in a second nucleic acid, divided by the total number ofnucleobases in the first nucleic acid. Percent identity (or percentcomplementarity) between particular stretches of nucleotide sequenceswithin nucleic acid molecules or amino acid sequences withinpolypeptides can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs (Altschul et al.,J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997,7, 649-656) or by using the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).Herein, if reference is made to percent sequence identity, the higherpercentages of sequence identity are preferred over the lower ones.

As used herein, “substantially identical” used herein may mean that afirst and second nucleobase sequence are at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 98% at least 99%, or 100%, identicalover a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, or 40 or more nucleobases.

As used herein, “hybridize” means the annealing of complementary nucleicacids that occurs through nucleobase complementarity.

As used herein, “mismatch” means a nucleobase of a first nucleic acidthat is not capable of pairing with a nucleobase at a correspondingposition of a second nucleic acid.

As used herein, “identical” means having the same nucleobase sequence.

As used herein, “hsa-miR-6883-5p” means the modified oligonucleotidehaving the nucleobase sequence set forth in SEQ ID NO: 1.

As used herein, “hsa-miR-149-3p” means the modified oligonucleotidehaving the nucleobase sequence set forth in SEQ ID NO: 2.

As used herein, “hsa-miR-6785-5p” means the modified oligonucleotidehaving the nucleobase sequence set forth in SEQ ID NO: 3.

As used herein, “hsa-miR-4728-5p” means the modified oligonucleotidehaving the nucleobase sequence set forth in SEQ ID NO: 4.

As used herein, “miRNA” or “miR” means a non-coding RNA from about 18 toabout nucleobases in length.

As used herein, “oligomeric compound” means a compound comprising apolymer of linked monomeric subunits.

As used herein, “oligonucleotide” means a polymer of linked nucleosides,each of which can be modified or unmodified, independent from oneanother.

As used herein, “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage between nucleosides.

As used herein, “natural sugar” means a sugar found in DNA (2′-H) or RNA(2′-OH).

As used herein, “natural nucleobase” means a nucleobase that isunmodified relative to its naturally occurring “internucleoside linkage”means a covalent linkage between adjacent nucleosides.

As used herein, “linked nucleosides” means nucleosides joined by acovalent linkage.

As used herein, “nucleobase” means a heterocyclic moiety capable ofnon-covalently pairing with another nucleobase.

As used herein, “nucleoside” means a nucleobase linked to a sugar.

As used herein, “nucleotide” means a nucleoside having a phosphate groupcovalently linked to the sugar portion of a nucleoside.

As used herein, “modified oligonucleotide” means an oligonucleotidehaving one or more modifications relative to a naturally occurringterminus, sugar, nucleobase, and/or internucleoside linkage.

As used herein, “modified internucleoside linkage” means any change froma naturally occurring internucleoside linkage.

As used herein, “phosphorothioate internucleoside linkage” means alinkage between nucleosides where one of the non-bridging atoms is asulfur atom.

As used herein, “modified sugar” means substitution and/or any changefrom a natural sugar.

As used herein, “modified nucleobase” means any substitution and/orchange from a natural nucleobase.

As used herein, “5-methylcytosine” means a cytosine modified with amethyl group attached to the 5′ position.

As used herein, “2′-O-methyl sugar” or “2′-0-Me sugar” means a sugarhaving an O-methyl modification at the 2′ position.

As used herein, “2′-O-methoxyethyl sugar” or “2′-MOE sugar” means asugar having a O-methoxyethyl modification at the 2′ position.

As used herein, “2′-0-fluoro” or “2′-F” means a sugar having a fluoromodification of the 2′ position.

As used herein, “bicyclic sugar moiety” means a sugar modified by thebridging of two non-geminal ring atoms.

As used herein, “2′-O-methoxyethyl nucleoside” means a 2′-modifiednucleoside having a 2′-O-methoxyethyl sugar modification.

As used herein, “2′-fluoro nucleoside” means a 2′-modified nucleosidehaving a 2′-fluoro sugar modification.

As used herein, “2′-O-methyl” nucleoside means a 2′-modified nucleosidehaving a 2′-O-methyl sugar modification.

As used herein, “bicyclic nucleoside” means a 2′-modified nucleosidehaving a bicyclic sugar moiety.

As used herein, “motif” means a pattern of modified and/or unmodifiednucleobases, sugars, and/or internucleoside linkages in anoligonucleotide.

As used herein, a “fully modified oligonucleotide” means eachnucleobase, each sugar, and/or each internucleoside linkage is modified.

As used herein, a “uniformly modified oligonucleotide” means eachnucleobase, each sugar, and/or each internucleoside linkage has the samemodification throughout the modified oligonucleotide.

As used herein, a “stabilizing modification” means a modification to anucleoside that provides enhanced stability to a modifiedoligonucleotide, in the presence of nucleases, relative to that providedby 2′-deoxynucleosides linked by phosphodiester internucleosidelinkages. For example, in some embodiments, a stabilizing modificationis a stabilizing nucleoside modification. In some embodiments, astabilizing modification is a internucleoside linkage modification.

As used herein, a “stabilizing nucleoside” means a nucleoside modifiedto provide enhanced nuclease stability to an oligonucleotide, relativeto that provided by a 2′-deoxynucleoside. In one embodiment, astabilizing nucleoside is a 2′-modified nucleoside.

As used herein, a “stabilizing internucleoside linkage” means aninternucleoside linkage that provides enhanced nuclease stability to anoligonucleotide relative to that provided by a phosphodiesterinternucleoside linkage. In one embodiment, a stabilizinginternucleoside linkage is a phosphorothioate internucleoside linkage.

The present disclosure provides oligonucleotides, such as modifiedoligonucleotides, consisting of 15 to 40 linked nucleobases, or a saltthereof, wherein the oligonucleotide comprises a nucleobase sequencethat is at least 80% identical to a nucleobase sequence ofhsa-miR-6883-5p, hsa-miR-149-3p, hsa-miR-6785-5p, or hsa-miR-4728-5p. Insome embodiments, hsa-miR-6883-5p comprises the nucleobase sequenceagggagggugugguauggaugu (SEQ ID NO: 1). In some embodiments,hsa-miR-149-3p comprises the nucleobase sequence agggagggacgggggcugugc(SEQ ID NO: 2). In some embodiments, hsa-miR-6785-5p comprises thenucleobase sequence ugggagggcguggaugauggug (SEQ ID NO: 3). In someembodiments, hsa-miR-4728-5p comprises the nucleobase sequenceugggaggggagaggcagcaagca (SEQ ID NO: 4). In some embodiments, theoligonucleotide comprises a nucleobase sequence that is at least 85%identical to a nucleobase sequence of hsa-miR-6883-5p, hsa-miR-149-3p,hsa-miR-6785-5p, or hsa-miR-4728-5p. In some embodiments, theoligonucleotide comprises a nucleobase sequence that is at least 90%identical to a nucleobase sequence of hsa-miR-6883-5p, hsa-miR-149-3p,hsa-miR-6785-5p, or hsa-miR-4728-5p. In some embodiments, theoligonucleotide comprises a nucleobase sequence that is at least 95%identical to a nucleobase sequence of hsa-miR-6883-5p, hsa-miR-149-3p,hsa-miR-6785-5p, or hsa-miR-4728-5p.

In some embodiments, an oligonucleotide consists of 15 to 30 linkednucleobases. In some embodiments, an oligonucleotide consists of 19 to24 linked nucleobases. In some embodiments, an oligonucleotide consistsof 21 to 24 linked nucleobases. In some embodiments, the oligonucleotideconsists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 linked nucleobases. Insome embodiments, the oligonucleotide consists of 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 linked nucleobases. Insome embodiments, the oligonucleotide consists of 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 linked nucleobases. In some embodiments, theoligonucleotide comprises a nucleobase sequence comprising at least 16,at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, or at least 23 contiguous nucleobases of a nucleobase sequenceof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In each ofthese embodiments, the oligonucleotide can be a modifiedoligonucleotide.

In some embodiments, the nucleobase sequence of the oligonucleotide hasno more than two mismatches compared to a nucleobase sequence selectedfrom SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In someembodiments, the nucleobase sequence of the oligonucleotide has no morethan one mismatch compared to a nucleobase sequence selected from SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In someembodiments, the nucleobase sequence of the oligonucleotide has onemismatch compared to a nucleobase sequence selected from SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In some embodiments, thenucleobase sequence of the oligonucleotide has no mismatches compared toa nucleobase sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, and SEQ ID NO: 4. In each of these embodiments, theoligonucleotide can be a modified oligonucleotide.

Suitable nucleic acids include, but are not limited to, deoxyribonucleicacid (DNA), ribonucleic acid (RNA), modified DNA or RNA, peptide nucleicacid (PNA), morpholino, locked nucleic acid (LNA), glycol nucleic acid(GNA), threose nucleic acid (TNA), DNA containing phosphorothioateresidues (S-oligos) and derivatives thereof, or any combination thereof.

In some embodiments, one or more additional nucleobases may be added toeither or both of the 3′ terminus and 5′ terminus of an oligonucleotidein comparison to the nucleobases sequences set forth in SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In some embodiments, theone or more additional linked nucleobases are at the 3′ terminus. Insome embodiments, the one or more additional linked nucleosides are atthe 5′ terminus. In some embodiments, two additional linked nucleosidesare linked to a terminus. In some embodiments, one additional nucleosideis linked to a terminus. In each of these embodiments, theoligonucleotide can be a modified oligonucleotide.

In some embodiments, the oligonucleotide comprises one or more modifiedinternucleoside linkages, modified sugars, or modified nucleobases, orany combination thereof. The nucleobase sequences set forth herein,including but not limited to those found in the Examples and in thesequence listing, are independent of any modification to the nucleicacid. As such, nucleic acids defined by a SEQ ID NO: may comprise,independently, one or more modifications to one or more sugar moieties,to one or more internucleoside linkages, and/or to one or morenucleobases. A modified nucleobase, sugar, and/or internucleosidelinkage may be selected over an unmodified form because of desirableproperties such as, for example, enhanced cellular uptake, enhancedaffinity for other oligonucleotides or nucleic acid targets andincreased stability in the presence of nucleases.

In some embodiments, at least one internucleoside linkage is a modifiedinternucleoside linkage. In some embodiments, each internucleosidelinkage is a modified internucleoside linkage. In some embodiments, amodified internucleoside linkage comprises a phosphorus atom. In someembodiments, a modified oligonucleotide comprises at least onephosphorothioate internucleoside linkage. In some embodiments, eachinternucleoside linkage of a modified oligonucleotide is aphosphorothioate internucleoside linkage. In some embodiments, amodified internucleoside linkage does not comprise a phosphorus atom. Insome such embodiments, an internucleoside linkage is formed by a shortchain alkyl internucleoside linkage. In some such embodiments, aninternucleoside linkage is formed by a cycloalkyl internucleosidelinkages. In some such embodiments, an internucleoside linkage is formedby a mixed heteroatom and alkyl internucleoside linkage. In some suchembodiments, an internucleoside linkage is formed by a mixed heteroatomand cycloalkyl internucleoside linkages. In some such embodiments, aninternucleoside linkage is formed by one or more short chainheteroatomic internucleoside linkages. In some such embodiments, aninternucleoside linkage is formed by one or more heterocyclicinternucleoside linkages. In some such embodiments, an internucleosidelinkage has an amide backbone. In some such embodiments, aninternucleoside linkage has mixed N, O, S and CH₂ component parts.

In some embodiments, at least one nucleobase of the modifiedoligonucleotide comprises a modified sugar. In some embodiments, each ofa plurality of nucleosides comprises a modified sugar. In someembodiments, each nucleoside of the modified oligonucleotide comprises amodified sugar. In each of these embodiments, the modified sugar may bea 2′-O-methoxyethyl sugar, a 2′-fluoro sugar, a 2′-O-methyl sugar, or abicyclic sugar moiety. In some embodiments, each of a plurality ofnucleosides comprises a 2′-O-methoxyethyl sugar and each of a pluralityof nucleosides comprises a 2′-fluoro sugar.

In some embodiments, the sugar-modified nucleosides can further comprisea natural or modified heterocyclic base moiety and/or a natural ormodified internucleoside linkage and may include further modificationsindependent from the sugar modification. In some embodiments, a sugarmodified nucleoside is a 2′-modified nucleoside, wherein the sugar ringis modified at the 2′ carbon from natural ribose or 2′-deoxyribose.

In some embodiments, a 2′-modified nucleoside has a bicyclic sugarmoiety. In some such embodiments, the bicyclic sugar moiety is a D sugarin the alpha configuration. In some such embodiments, the bicyclic sugarmoiety is a D sugar in the beta configuration. In some such embodiments,the bicyclic sugar moiety is an L sugar in the alpha configuration. Insome such embodiments, the bicyclic sugar moiety is an L sugar in thebeta configuration. In some embodiments, the bicyclic sugar moietycomprises a bridge group between the 2′ and the 4′-carbon atoms. In somesuch embodiments, the bridge group comprises from 1 to 8 linkedbiradical groups. In some embodiments, the bicyclic sugar moietycomprises from 1 to 4 linked biradical groups. In some embodiments, thebicyclic sugar moiety comprises 2 or 3 linked biradical groups. In someembodiments, the bicyclic sugar moiety comprises 2 linked biradicalgroups. Biradical groups are well known in the art.

In some embodiments, the modified oligonucleotide comprises at least onemodified nucleobase. In some embodiments, the modified nucleobase isselected from 5-hydroxymethyl cytosine, 7-deazaguanine and7-deazaadenine. In some embodiments, the modified nucleobase is selectedfrom 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.In some embodiments, the modified nucleobase is selected from5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2 aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. In some embodiments, the modified nucleobase isa 5-methylcytosine. In some embodiments, at least one nucleosidecomprises a cytosine, wherein the cytosine is a 5-methylcytosine. Insome embodiments, each cytosine is a 5-methylcytosine.

In some embodiments, a 2′-modified nucleoside comprises a 2′-substituentgroup selected from halo, allyl, amino, azido, —SH, —CN, —OCN, —CF₃,—OCF₃, —O—, —S—, or —N(R_(m))-alkyl; —O—, —S—, or —N(R_(m))-alkenyl;—O—, —S— or —N(R_(m))-alkynyl; —O-alkyleny-O-alkyl, alkynyl, alkaryl,aralkyl, —O-alkaryl, —O-aralkyl, —O(CH₂)₂SCH₃,—O—(CH₂)₂—O—N(R_(m))(R_(n)) or —O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H, an amino protecting group orsubstituted or unsubstituted C₁₋₁₀alkyl. These 2′-substituent groups canbe further substituted with one or more substituent groups independentlyselected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro,thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In some embodiments, a 2′-modified nucleoside comprises a 2′-substituentgroup selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂, CH₂—CH═CH₂,O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)),—O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substituted acetamide(O—CH₂—C(═O)—N(Rm)(R_(n)) where each R_(m) and R_(n) is, independently,H, an amino protecting group or substituted or unsubstituted C₁₋₁₀alkyl.In some embodiments, a 2′-modified nucleoside comprises a 2′-substituentgroup selected from F, OCF₃, O—CH₃, OCH₂CH₂OCH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, and O—CH₂—C(═O)—N(H)CH₃.

In some embodiments, a 2′-modified nucleoside comprises a 2′-substituentgroup selected from F, O—CH₃, and OCH₂CH₂OCH₃. In some embodiments, asugar-modified nucleoside is a 4′-thio modified nucleoside. In someembodiments, a sugar-modified nucleoside is a 4′-thio-2′-modifiednucleoside. A 4′-thio modified nucleoside has a B-D-ribonucleoside wherethe 4′-O replaced with 4′-S. A 4′-thio-2′-modified nucleoside is a4′-thio modified nucleoside having the 2′-OH replaced with a2′-substituent group. Suitable 2′-substituent groups include 2′-OCH₃,2-O—(CH₂)₂—OCH₃, and 2′-F.

In some embodiments, a modified nucleobase comprises a polycyclicheterocycle. In some embodiments, a modified nucleobase comprises atricyclic heterocycle. In some embodiments, a modified nucleobasecomprises a phenoxazine derivative. In some embodiments, the phenoxazinecan be further modified to form a nucleobase known in the art as aG-clamp.

In some embodiments, the oligonucleotide compound comprises a modifiedoligonucleotide conjugated to one or more moieties which enhance theactivity, cellular distribution or cellular uptake of the resultingantisense oligonucleotides. In some such embodiments, the moiety is acholesterol moiety or a lipid moiety. Additional moieties forconjugation include carbohydrates, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. In some embodiments, a conjugate groupis attached directly to a modified oligonucleotide. In some embodiments,a conjugate group is attached to a modified oligonucleotide by a linkingmoiety selected from amino, hydroxyl, carboxylic acid, thiol,unsaturations (e.g., double or triple bonds), 8-amino-3,6-dioxaoctanoicacid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted C₁₋₁₀alkyl,substituted or unsubstituted C₂₋₁₀alkenyl, and substituted orunsubstituted C₂₋₁₀alkynyl. In some such embodiments, a substituentgroup is selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl,nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In some such embodiments, the oligonucleotide compound comprises amodified oligonucleotide having one or more stabilizing groups that areattached to one or both termini of a modified oligonucleotide to enhanceproperties such as, for example, nuclease stability. Included instabilizing groups are cap structures. These terminal modificationsprotect a modified oligonucleotide from exonuclease degradation, and canhelp in delivery and/or localization within a cell. The cap can bepresent at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), orcan be present on both termini Cap structures include, for example,inverted deoxy abasic caps.

Additional cap structures include, but are not limited to, a4′,5′-methylene nucleotide, a 1-(beta-D-erythrofuranosyl) nucleotide, a4′-thio nucleotide, a carbocyclic nucleotide, a 1,5-anhydrohexitolnucleotide, an L-nucleotide, an alpha-nucleotide, a modified basenucleotide, a phosphorodithioate linkage, a threopentofuranosylnucleotide, an acyclic 3′,4′-seco nucleotide, an acyclic3,4-dihydroxybutyl nucleotide, an acyclic 3,5-dihydroxypentylnucleotide, a 3′-3′-inverted nucleotide moiety, a 3′-3′-inverted abasicmoiety, a 3′-2′-inverted nucleotide moiety, a 3′-2′-inverted abasicmoiety, a 1,4-butanediol phosphate, a 3′-phosphoramidate, ahexylphosphate, an aminohexyl phosphate, a 3′-phosphate, a3′-phosphorothioate, a phosphorodithioate, a bridging methylphosphonatemoiety, and a non-bridging methylphosphonate moiety 5′-amino-alkylphosphate, a 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, a6-aminohexyl phosphate, a 1,2-aminododecyl phosphate, a hydroxypropylphosphate, a 5′-5′-inverted nucleotide moiety, a 5′-5′-inverted abasicmoiety, a 5′-phosphoramidate, a 5′-phosphorothioate, a 5′-amino, abridging and/or non-bridging 5′-phosphoramidate, a phosphorothioate, anda 5′-mercapto moiety.

The present disclosure also provides pharmaceutical compositionscomprising one or more of the oligonucleotides described herein. In someembodiments, the oligonucleotide consists of 15 to 30 linkednucleosides, or a salt thereof, wherein the modified oligonucleotidecomprises a nucleobase sequence that is at least 80% identical to anucleobase sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, and a pharmaceutically acceptable carrier or diluent.In each of these embodiments, the oligonucleotide can be a modifiedoligonucleotide.

In some embodiments, the compositions may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions. Theformulations can be sterilized and, if desired, mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, colorings,flavorings and/or aromatic substances and the like which do notdeleteriously interact with the oligonucleotide(s) of the formulation.

In some embodiments, pharmaceutical compositions comprise one or moremodified oligonucleotides and one or more excipients. In some suchembodiments, excipients are selected from water, salt solutions,alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesiumstearate, talc, silicic acid, viscous paraffin, hydroxymethylcelluloseand polyvinylpyrrolidone.

In some embodiments, a pharmaceutical composition is prepared usingknown techniques, including, but not limited to mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or tab letting processes.

In some embodiments, a pharmaceutical composition is a liquid (e.g., asuspension, elixir and/or solution). In some such embodiments, a liquidpharmaceutical composition is prepared using ingredients known in theart, including, but not limited to, water, glycols, oils, alcohols,flavoring agents, preservatives, and coloring agents.

In some embodiments, a pharmaceutical composition is a solid (e.g., apowder, tablet, and/or capsule). In some such embodiments, a solidpharmaceutical composition comprising one or more oligonucleotides isprepared using ingredients known in the art, including, but not limitedto, starches, sugars, diluents, granulating agents, lubricants, binders,and disintegrating agents.

In some embodiments, a pharmaceutical composition is formulated as adepot preparation. Some such depot preparations are typically longeracting than non-depot preparations. In some embodiments, suchpreparations are administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Insome embodiments, depot preparations are prepared using suitablepolymeric or hydrophobic materials (for example an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In some embodiments, a pharmaceutical composition comprises a deliverysystem. Examples of delivery systems include, but are not limited to,liposomes and emulsions. Delivery systems are useful for preparingpharmaceutical compositions including those comprising hydrophobiccompounds. In some embodiments, some organic solvents such asdimethylsulfoxide are used. In some embodiments, presently availableRNAi packaging technology can be used to packing the miRNA in lipidcomplexes and to deliver the miRNA. The delivery system can alsocomprise nanoparticules or nano-complexes. The delivery system can alsocomprise bacterial mini-cells comprising RNA duplexes.

In some embodiments, a pharmaceutical composition comprises one or moretissue-specific delivery molecules designed to deliver the one or morepharmaceutical agents to specific tissues or cell types. For example, insome embodiments, pharmaceutical compositions include liposomes coatedwith a tissue-specific antibody.

In some embodiments, a pharmaceutical composition comprises a cosolventsystem. Some such co-solvent systems comprise, for example, benzylalcohol, a nonpolar surfactant, a water-miscible organic polymer, and anaqueous phase. In some embodiments, such cosolvent systems are used forhydrophobic compounds. A non-limiting example of such a co-solventsystem is the VPD co-solvent system, which is a solution of absoluteethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolarsurfactant Polysorbate 80™ and 65% w/v polyethylene glycol300. Theproportions of such co-solvent systems may be varied considerablywithout significantly altering their solubility and toxicitycharacteristics. Furthermore, the identity of co-solvent components maybe varied: for example, other surfactants may be used instead ofPolysorbate 80™; the fraction size of polyethylene glycol may be varied;other biocompatible polymers may replace polyethylene glycol, e.g.,polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In some embodiments, a pharmaceutical composition comprises asustained-release system. A non-limiting example of such asustained-release system is a semi-permeable matrix of solid hydrophobicpolymers. In some embodiments, sustained-release systems may, dependingon their chemical nature, release pharmaceutical agents over a period ofhours, days, weeks or months.

In some embodiments, a pharmaceutical composition is prepared for oraladministration. In some such embodiments, a pharmaceutical compositionis formulated by combining one or more compounds comprising any one ormore of the oligonucleotides described herein with one or morepharmaceutically acceptable carriers. Some such carriers enablepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a subject. In some embodiments, pharmaceuticalcompositions for oral use are obtained by mixing oligonucleotide and oneor more solid excipient. Suitable excipients include, but are notlimited to, fillers, such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In someembodiments, such a mixture is optionally ground and auxiliaries areoptionally added. In some embodiments, pharmaceutical compositions areformed to obtain tablets or dragee cores. In some embodiments,disintegrating agents (e.g., cross-linked polyvinylpyrrolidone, agar, oralginic acid or a salt thereof, such as sodium alginate) are added.

In some embodiments, dragee cores are provided with coatings. In somesuch embodiments, concentrated sugar solutions may be used, which mayoptionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopolgel, polyethylene glycol, and/or titanium dioxide, lacquer solutions,and suitable organic solvents or solvent mixtures. Dyestuffs or pigmentsmay be added to tablets or dragee coatings.

In some embodiments, pharmaceutical compositions for oral administrationare push-fit capsules made of gelatin. Some such push-fit capsulescomprise one or more of the oligonucleotides described herein inadmixture with one or more filler such as lactose, binders such asstarches, and/or lubricants such as talc or magnesium stearate and,optionally, stabilizers. In some embodiments, pharmaceuticalcompositions for oral administration are soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. In some softcapsules, one or more of the oligonucleotides described herein are bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycols. In addition, stabilizers maybe added.

In some embodiments, pharmaceutical compositions are prepared for buccaladministration. Some such pharmaceutical compositions are tablets orlozenges formulated in conventional manner.

In some embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, intraperitoneal,subcutaneous, intramuscular, etc.). In some such embodiments, apharmaceutical composition comprises a carrier and is formulated inaqueous solution, such as water or physiologically compatible bufferssuch as Hanks's solution, Ringer's solution, or physiological salinebuffer. In some embodiments, other ingredients are included (e.g.,ingredients that aid in solubility or serve as preservatives). In someembodiments, injectable suspensions are prepared using appropriateliquid carriers, suspending agents and the like. Some pharmaceuticalcompositions for injection are presented in unit dosage form, e.g., inampoules or in multi-dose containers. Some pharmaceutical compositionsfor injection are suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Some solvents suitable for use inpharmaceutical compositions for injection include, but are not limitedto, lipophilic solvents and fatty oils, such as sesame oil, syntheticfatty acid esters, such as ethyl oleate or triglycerides, and liposomes.Aqueous injection suspensions may contain substances that increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, such suspensions may also containsuitable stabilizers or agents that increase the solubility of theoligonucleotides described herein to allow for the preparation of highlyconcentrated solutions. In some embodiments, a pharmaceuticalcomposition is prepared for transmucosal administration. In some suchembodiments penetrants appropriate to the barrier to be permeated areused in the formulation. Such penetrants are generally known in the art.

In some embodiments, a pharmaceutical composition is prepared foradministration by inhalation. Some such pharmaceutical compositions forinhalation are prepared in the form of an aerosol spray in a pressurizedpack or a nebulizer. Some such pharmaceutical compositions comprise apropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In someembodiments using a pressurized aerosol, the dosage unit may bedetermined with a valve that delivers a metered amount. In someembodiments, capsules and cartridges for use in an inhaler orinsufflator may be formulated. Some such formulations comprise a powdermixture of one or more of the oligonucleotides described herein and asuitable powder base such as lactose or starch.

In some embodiments, a pharmaceutical composition is prepared for rectaladministration, such as a suppositories or retention enema. Some suchpharmaceutical compositions comprise known ingredients, such as cocoabutter and/or other glycerides.

In some embodiments, a pharmaceutical composition is prepared fortopical administration. Some such pharmaceutical compositions comprisebland moisturizing bases, such as ointments or creams. Exemplarysuitable ointment bases include, but are not limited to, petrolatum,petrolatum plus volatile silicones, and lanolin and water in oilemulsions. Exemplary suitable cream bases include, but are not limitedto, cold cream and hydrophilic ointment.

In some embodiments, a pharmaceutical composition comprises a modifiedoligonucleotide in a therapeutically effective amount. In someembodiments, the therapeutically effective amount is sufficient toprevent, alleviate or ameliorate symptoms of a disease or to prolong thesurvival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In some embodiments, the pharmaceutical composition may further compriseat least one additional therapeutic agent. The additional therapeuticagent may be a chemotherapeutic agent. In some embodiments, thechemotherapeutic agent is a platinum-based chemotherapeutic agent suchas, for example, cisplatin, carboplatin, oxaliplatin, nedaplatin,triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin. Insome embodiments, the chemotherapeutic agent is a taxane such as, forexample, paclitaxel, docetaxel, or cabazitaxel. In some embodiments, thechemotherapeutic agent is a type I topoisomerase inhibitor such as, forexample, irinotecan, topotecan, camptothecin, or lamellarin D. In someembodiments, the chemotherapeutic agent is a type II topoisomeraseinhibitor such as, for example, etoposide (VP-16), teniposide,doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticines,aurintricarboxylic acid, or HU-331. In some embodiments, thechemotherapeutic agent is a combination of chemotherapeutic agents suchas, for example, CHOP (cyclophosphamide, doxorubicin(hydroxydaunomycin), vincristine (Oncovie®), and prednisolone). In someembodiments, the chemotherapeutic agent may be selected from5-fluorouracil, cisplatin, gemcitabine, doxorubicine, mitomycin c,sorafenib, etoposide, carboplatin, epirubicin, irinotecan andoxaliplatin. In some embodiments, the chemotherapeutic is 5-fluorouracilor irinotecan. The pharmaceutical composition may comprise one or moreof the oligonucleotide compounds described herein in combination withone or more of the additional therapeutic agents. For example, thepharmaceutical composition may comprise an one of more oligonucleotidesconsisting of 15 to 40 linked nucleobases, or a salt thereof, whereinthe oligonucleotide comprises a nucleobase sequence that is at least 80%identical to a nucleobase sequence of hsa-miR-6883-5p, hsa-miR-149-3p,hsa-miR-6785-5p, or hsa-miR-4728-5p, in combination with any one or moreof 5-fluorouracil, cisplatin, gemcitabine, doxorubicine, mitomycin c,sorafenib, etoposide, carboplatin, epirubicin, irinotecan andoxaliplatin. In some embodiments, the pharmaceutical compositioncomprises an one of more oligonucleotides consisting of 15 to 40 linkednucleobases, or a salt thereof, wherein the oligonucleotide comprises anucleobase sequence that is at least 80% identical to a nucleobasesequence of hsa-miR-6883-5p, hsa-miR-149-3p, hsa-miR-6785-5p, orhsa-miR-4728-5p, in combination with 5-fluorouracil and/or irinotecan.

In some embodiments, an additional therapy may be a pharmaceutical agentthat enhances the body's immune system, including low-dosecyclophosphamide, thymostimulin, vitamins and nutritional supplements(e.g., antioxidants, including vitamins A, C, E, beta-carotene, zinc,selenium, glutathione, coenzyme Q-10 and echinacea), and vaccines, e.g.,the immunostimulating complex (ISCOM), which comprises a vaccineformulation that combines a multimeric presentation of antigen and anadjuvant.

In some embodiments, a pharmaceutical agent that induces the expressionof the miRNAs disclosed herein, or induces the expression of PER1 orregulates the expression of PER1, such as atypical psychotics including,but not limited to, quetiapine and haloperidol can be used. In someembodiments, the pharmaceutical agent is melatonin. In some embodiments,the pharmaceutical agent for inducing the expression of the miRNAs orPER1 is forskolin, interleukin-6, or Sp-5,6-DCI-cBiMPS. Thesepharmaceutical agents may be present in a pharmaceutical composition.

The present disclosure also provides methods for treating a tumor orcancer, comprising administering to a subject in need thereof one ormore of the oligonucleotides described herein, and/or a pharmaceuticalagent that induces the production of the one or more oligonucleotidesand/or induces PER1 expression. In some embodiments, the oligonucleotideconsists of 15 to 30 linked nucleosides, wherein the oligonucleotidecomprises a nucleobase sequence that is at least 80% identical to anucleobase sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, and SEQ ID NO: 4. In some embodiments, the oligonucleotide is amodified oligonucleotide as described herein.

The present disclosure also provides methods for treating a tumor orcancer, comprising administering to a subject in need thereof apharmaceutical composition comprising one or more of theoligonucleotides described herein, or a pharmaceutical agent thatinduces the production of the one or more oligonucleotides and/orinduces PER1 expression. In some embodiments, the oligonucleotideconsists of 15 to 30 linked nucleosides, wherein the oligonucleotidecomprises a nucleobase sequence that is at least 80% identical to anucleobase sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, and SEQ ID NO: 4. In some embodiments, the oligonucleotide is amodified oligonucleotide as described herein.

In some embodiments, the cancer being treated is a tumor or solid tumor.In some embodiments, the cancer cell or tumor overexpresses a CDK, suchas CDK4 and/or CDK6. In some embodiments, the cancer cell or tumorexhibits a loss of p16. In some embodiments, the cancer cell or tumorexhibits a loss of Rb. In some embodiments, the cancer cell or tumorexhibits overexpression of cyclin D1. In some embodiments, the cancercell or tumor is in need of inhibition of a CDK, such as CDK4 and/orCDK6. In some embodiments, the tumor or cancer is pancreatic, melanoma,colorectal, colon, lung, breast, or leukemia. In some embodiments, thecancer is colon cancer, colorectal cancer, lung cancer, or melanoma. Insome embodiments, the cancer is colon cancer. In some embodiments, thecancer is pancreatic cancer. In some embodiments, the cancer iscolorectal cancer. In some embodiments, the cancer is lung cancer. Insome embodiments, the cancer is melanoma. In some embodiments, thetumors may be less well-oxygenated than the normal tissues from whichthey arose (i.e., “tumor hypoxia”) which, in some cases, may lead toresistance to radiotherapy and anticancer chemotherapy as well aspredisposing for increased tumor metastases.

In some embodiments, the methods described herein use one or moreoligonucleotides or modified oligonucleotides that is/are targeted toCDK4 and/or CDK6. The oligonucleotides or modified oligonucleotides canbe administered with or without being integrated into a vector. Theoligonucleotides or modified oligonucleotides can also be used in theform of double stranded entities, whereby the appropriate strand isproduced inside a cell.

In some embodiments, administration of a compound comprises intravenousadministration, subcutaneous administration, intratumoraladministration, intraperitoneal administration, or chemoembolization.

In some embodiments, the methods further comprise administering at leastone additional therapy. The additional therapy may be a chemotherapeuticagent. The chemotherapeutic agent may be selected from 5-fluorouracil,cisplatin, gemcitabine, doxorubicine, mitomycin c, sorafenib, etoposide,carboplatin, epirubicin, irinotecan and oxaliplatin. In someembodiments, the chemotherapeutic is 5-fluorouracil or irinotecan. Theadditional therapy may be administered at the same time, lessfrequently, or more frequently than a compound or pharmaceuticalcomposition described herein. In some embodiments, the additionaltherapy is surgical resection and/or chemoembolization.

In some embodiments, any one or more of the oligonucleotides describedherein is administered at a dose selected from 50, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mg. Theoligonucleotide may be administered one per day, once per week, once pertwo weeks, once per three weeks, or once per four weeks.

In some embodiments, the administration of a compound results inreduction of tumor size and/or tumor number. In some embodiments, theadministration of a compound prevents an increase in tumor size and/ortumor number. In some embodiments, the administration of a compoundprevents, slows, and/or stops metastatic progression. In someembodiments, the administration of a compound extends the overallsurvival time of the subject. In some embodiments, the administration ofa compound extends the progression-free survival of the subject. In someembodiments, administration of a compound prevents the recurrence oftumors. In some embodiments, administration of a compound preventsrecurrence of tumor metastasis.

A subject may be diagnosed with a tumor or cancer, such as colon cancer,following the administration of medical tests well known to those in themedical profession. The diagnosis of a tumor or cancer, such as coloncancer, can be made by imaging tests such as ultrasound, helicalcomputed tomography (CT) scan, triple phase CT scan, or magneticresonance imaging (MRI). The imaging tests allow the assessment of thetumor size, number, location, metastasis, patency and/or invasion ofadjacent tissue by the tumor. This assessment aids the decision as tothe mode of therapeutic or palliative intervention that is appropriate.The final diagnosis is typically confirmed by needle biopsy andhistopathological examination.

Administration of a pharmaceutical composition to a subject having atumor can result in one or more clinically desirable outcomes. Suchclinically desirable outcomes include reduction of tumor number orreduction of tumor size. Additional clinically desirable outcomesinclude the extension of overall survival time of the subject, and/orextension of progression-free survival time of the subject. In someembodiments, administration of a pharmaceutical composition prevents anincrease in tumor size and/or tumor number. In some embodiments,administration of a pharmaceutical composition prevents metastaticprogression. In some embodiments, administration of a pharmaceuticalcomposition slows or stops metastatic progression. In some embodiments,administration of a pharmaceutical composition prevents the recurrenceof tumors. In some embodiments, administration of a pharmaceuticalcomposition prevents recurrence of tumor metastasis. In someembodiments, administration of a pharmaceutical composition prevents therecurrence of tumors. Administration of a pharmaceutical composition totumor cells may result in desirable phenotypic effects. In someembodiments, an oligonucleotide may stop, slow or reduce theuncontrolled proliferation of tumor cells. In some embodiments, anoligonucleotide may induce apoptosis in tumor cells. In someembodiments, an oligonucleotide may reduce tumor cell survival.

In some embodiments, one or more pharmaceutical compositions and one ormore other pharmaceutical agents are administered at the same time. Insome embodiments, one or more pharmaceutical compositions and one ormore other pharmaceutical agents are administered at different times. Insome embodiments, one or more pharmaceutical compositions and one ormore other pharmaceutical agents are prepared together in a singleformulation. In some embodiments, one or more pharmaceuticalcompositions and one or more other pharmaceutical agents are preparedseparately.

In some embodiments, a pharmaceutical composition is administered in theform of a dosage unit (e.g., tablet, capsule, bolus, etc.). In someembodiments, such pharmaceutical compositions comprise any one or moreof the oligonucleotides or modified oligonucleotides described herein ina dose selected from 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 270 mg, 280 mg, 285 mg, 290mg, 295 mg, 300 mg, 305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335mg, 340 mg, 345 mg, 350 mg, 355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380mg, 385 mg, 390 mg, 395 mg, 400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425mg, 430 mg, 435 mg, 440 mg, 445 mg, 450 mg, 455 mg, 460 mg, 465 mg, 470mg, 475 mg, 480 mg, 485 mg, 490 mg, 495 mg, 500 mg, 505 mg, 510 mg, 515mg, 520 mg, 525 mg, 530 mg, 535 mg, 540 mg, 545 mg, 550 mg, 555 mg, 560mg, 565 mg, 570 mg, 575 mg, 580 mg, 585 mg, 590 mg, 595 mg, 600 mg, 605mg, 610 mg, 615 mg, 620 mg, 625 mg, 630 mg, 635 mg, 640 mg, 645 mg, 650mg, 655 mg, 660 mg, 665 mg, 670 mg, 675 mg, 680 mg, 685 mg, 690 mg, 695mg, 700 mg, 705 mg, 710 mg, 715 mg, 720 mg, 725 mg, 730 mg, 735 mg, 740mg, 745 mg, 750 mg, 755 mg, 760 mg, 765 mg, 770 mg, 775 mg, 780 mg, 785mg, 790 mg, 795 mg, and 800 mg. In some such embodiments, apharmaceutical composition comprises a dose of modified oligonucleotideselected from 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, and 800 mg.

In some embodiments, a pharmaceutical agent is sterile lyophilizedoligonucleotide that is reconstituted with a suitable diluent, e.g.,sterile water for injection or sterile saline for injection. Thereconstituted product is administered as a subcutaneous injection or asan intravenous infusion after dilution into saline. The lyophilized drugproduct consists of any one or more of the oligonucleotides or modifiedoligonucleotides described herein which has been prepared in water forinjection, or in saline for injection, adjusted to pH 7.0-9.0 with acidor base during preparation, and then lyophilized. The lyophilizedmodified oligonucleotide may be 25-800 mg of any one or more of theoligonucleotides or modified oligonucleotides described herein. It isunderstood that this encompasses 25, 50, 75, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 425, 450, 475, 500, 525, 550, 575,600, 625, 650, 675, 700, 725, 750, 775, and 800 mg of modifiedlyophilized oligonucleotide. The lyophilized drug product may bepackaged in a 2 mL Type I, clear glass vial (ammonium sulfate-treated),stoppered with a bromobutyl rubber closure and sealed with an aluminumFLIP-OFF® overseal.

The present disclosure also provides methods of detecting and/ordetermining the level of any one or more of the miRNA described herein.The detection and/or level determination can be carried out byconventional means known in the art. The level of particular miRNAs canbe used as disease progress markers for any of the cancers disclosedherein. The miRNAs can also be used to predict and/or monitor atherapeutic response.

The present disclosure also provides any one or more of theoligonucleotide compounds described herein, or compositions comprisingthe same, for use in treating or preventing cancer or tumors.

The present disclosure also provides any one or more of theoligonucleotide compounds described herein, or compositions comprisingthe same, for use in the manufacture of a medicament for treating orpreventing cancer or tumors.

The present disclosure also provides uses of any one or more of theoligonucleotide compounds described herein, or compositions comprisingthe same, for treating or preventing cancer or tumors.

The present disclosure also provides uses of any one or more of theoligonucleotide compounds described herein, or compositions comprisingthe same, in the manufacture of a medicament for treating or preventingcancer or tumors.

The present disclosure also provides any one or more of theoligonucleotide compounds described herein, or compositions comprisingthe same, or methods of preparing the same, or methods of using thesame, or uses any one or more of the oligonucleotide compounds describedherein, or compositions comprising the same, substantially as describedwith reference to the accompanying examples and/or figures.

In order that the subject matter disclosed herein may be moreefficiently understood, examples are provided below. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting the claimed subject matter in anymanner. Throughout these examples, molecular cloning reactions, andother standard recombinant DNA techniques, were carried out according tomethods described in Maniatis et al., Molecular Cloning—A LaboratoryManual, 2nd ed., Cold Spring Harbor Press (1989), using commerciallyavailable reagents, except where otherwise noted.

EXAMPLES Example 1: General Materials and Methods

Cell culture and reagents: All colorectal, pancreatic and melanoma celllines were obtained from American Type Culture Collection and maintainedin the recommended media. miRNA mimics for hsa-miR-6883-5p,hsa-miR-149*, and hsa-miR-206 (HMI2616, HMI0241 and HMI0364) werepurchased from Sigma-Aldrich. siRNA for CDK4 and CDK6 (sc-29261 andsc-29264) were purchased from Santa Cruz Biotechnology. TheCDK4-Luciferase construct was obtained from Origene Technologies.

Transfection of miRNA mimics, siRNA and plasmid constructs: All miRNAmimics and siRNA transfections were performed by reverse transfectionusing Lipofectamine RNAiMAX (Life technologies, Grand Island, N.Y.). 80nM siRNA was used in all experiments for HT-29, RKO and SW-480 celllines. 40 nM siRNA was used for HCT-116 cells. miRNA mimics weretransfected at concentrations of either 25 nM, 50 nM, or 100 nM, asindicated in respective assays. CDK4-Luciferase vector was transfectedin HCT-116 cells using Lipofectamine 3000 (Life technologies, GrandIsland, N.Y.) and stable cells were selected using G418 antibiotic (500μg/mL).

Luciferase assays: CDK4-Luciferase containing stable HCT-116 cells werereverse-transfected with either scramble duplex or 50 nM miRNA mimicsLuciferase signal was detected 48 hours post-transfection and RelativeLuciferase Units (RLU) were calculated by normalizing luciferase signalper μg of protein per assay well. All transfections were performed intriplicates and reported as RLU units±SEM.

Cell Proliferation assays: Five thousand to ten thousand cells werereverse transfected with either scramble duplex or miRNA to a netconcentration of 50 nM and plated in 96-well plate. Cell viability wasmeasured 72 hours post-transfection using CellTiter-Glo® LuminescentCell Viability Assay (Promega). Percent cell viability was calculated bynormalizing the luminescence signal to scramble duplex wells. Alltransfections were performed in triplicates and reported as %Viability±SEM.

Cell Cycle Analysis: All cell lines were reverse transfected with eitherscramble duplex or miRNA mimic. At 72 hours post-transfection, bothfloating and adherent cells were collected and fixed in 70% ethanol,followed by RNase A treatment and PI staining. Cell death (sub-G1) wasquantified by propidium iodide (PI) staining and fluorescence-activatedcell sorting (FACS). Flo-Jo analysis was performed to quantify thedistribution of cells in G1, S, and G2-M phases of cell cycle underdifferent transfection conditions.

Colony formation assays: A total of 0.1×10⁶ cells were reversetransfected with either scramble duplex or miRNA mimics to netconcentration of 50 nM (HT-29 and HCT-116) or 100 nM (RKO and SW-480)for 72 hours. At 72 hours, transfected cells were harvested and 500cells per treatment group were plated in triplicate in 6-well plates forcolony formation. Colonies were crystal violet stained on Day 14,imaged, counted and reported as the number of colonies±SEM.

Quantitative RT-PCR (qRT-PCR): Total RNA, which includes miRNA, wasisolated using the Quick-RNA™ MiniPrep kit (Zymo Research, Irvine,Calif.). One μg of total RNA from each sample was subjected to cDNAsynthesis using SuperScript® III Reverse Transcriptase kit (LifeTechnologies, Grand Island, N.Y.), for detection of CDK4, CDK6, andhousekeeping genes. For detection of miRNAs, 0.5 μg of total RNA wasreverse transcribed using TaqMan® MicroRNA Reverse Transcription Kit(Life Technologies, Grand Island, N.Y.). The relative expression of thereported genes and miRNAs was determined using real-time PCR performedon Applied Biosystems 7900HT Fast Real-Time PCR system. GAPDH and RNU6Bwere used as the endogenous controls for mRNA and miRNA samplesrespectively. Each cDNA sample was amplified using Power SYBR Green(Applied Biosystems, CA) and miRNA components were quantified usingTaqMan® Universal Master Mix II, no UNG (Applied Biosystems, CA). TaqManmiRNA assays were purchased from Applied Biosystems and used as permanufacturer's instructions.

Western blot: Western blotting was performed by routine and well knownprocedures. The following antibodies were used: CDK4 (Santa CruzBiotechnology, sc-260), CDK6 (CST, D4S8S), CDK1 (Santa CruzBiotechnology, sc-54), Cyclin-D1 (CST, 92G2), p-Rb (S795) (CST, 9301S),Total Rb, BCLxL (CST, 2764S), PARP (CST, 9542), p53-DO1 (Santa CruzBiotechnology, sc-126), p21 (Calbiochem, OP64), and β-actin (Sigma,A5441).

Statistical analysis: Data are presented as the mean±standard error ofmean from at least three replicates. The Student's two-tailed t-test inGraphPad Prism was used for pairwise analysis. Statistically significantchanges (*p≤0.05, **p≤0.01 and ***≤0.001) are indicated.

Example 2: CDK4 and CDK6 are Important Therapeutic Targets and can beRegulated with miRNAs in CRC

Events of overexpression of cell cycle oncogenes (CDKs, Cyclins) andsuppression of tumor suppressors (p16, Rb) in the CDK4/6-Rb pathway aremutually exclusive and tumor-type specific. To assess the status ofthese events in CRC, RNA-sequencing data from The Cancer Genome Atlas(TCGA) for normal and tumor samples (T/N) was analyzed. Of the 50 T/Nsamples analyzed as shown in FIG. 1A, both CDK4 (left panel) and CDK6(right panel) expression were significantly high in the tumor samples(p=2.2 e-16 and p=1.594 e-08 respectively). There was no significantchange in the RNA levels of tumor suppressors p16 and Rb. These findingsare in consensus with previous reports which have shown overexpressionof CDK4 and CDK6 in IHC samples with differences on staging (Zhao etal., World J. Gastroenterol., 2003, 9, 2202-2206; and Zhao et al., WorldJ. Gastroenterol., 2006, 12, 6391-6396).

To determine whether CDK4 and CDK6 can be negatively regulated bymiRNAs, an in silico approach using TargetScan, an online computationalalgorithm, was taken to find miRNAs that could target the 3′UTR of bothCDK4 and CDK6. There have been prior reports of conserved miRNAs,including miR-206, miR-124-3p, and miR-15-5p, that examine CDK4targeting alone in melanoma and other tumor types. One goal was to focuson novel and uncharacterized miRNAs that also had relevance in CRC andcould target both CDK4 and CDK6. Based on a combination of TCGA analysisand TargetScan, a new family of miRNAs encompassing miRs 6883-5p, 149*,6785-5p and 4728-5p was developed. Each of these miRNAs were predictedto target both CDK4 and CDK6 3′UTR with 8-mer and 7-mer-1A binding sites(Table 1 and 2, respectively, of FIG. 1B). miRs 6883-5p and 149* werefurther examined for their novelty and relevance to CRC. As seen in FIG.1C, expression of miR-149* was significantly lost in 11 patient CRCtumors as compared to normal tissue (p=0.0049) as assayed by RNA-sequsing TCGA analysis. Loss of miR-149* was also correlative to thestaging of the tumor (data not shown). Each of the 11 patients also hadsignificant increase in CDK4 expression with little to no change in theother markers of the CDK4/6-Rb pathway (see, FIG. 1C). As formiR-6883-5p, no data was located in TCGA. The TCGA analysis showed thatRNA expression of PER1 is significantly lost in the same 50 T/N samplesassayed prior (see, FIG. 1D). Thus, the in silico analysis suggests thatrestoring expression of miRs 6883-5p and 149* can be used as therapiesin treating CRC.

Referring in particular to FIGS. 1A, 1B, 1C, and 1D, data is presentedshowing that CDK4 and CDK6 are important therapeutic targets in CRC.FIG. 1A shows RNA expression data from TCGA CRC patient samples showingexpression of CDK4 and CDK6 in 50 tumor and normal samples. Box plotsindicate the log_(in) RNA expression of normal samples compared to tumorsamples for every gene of interest. p-values were obtained from theWilcoxon test for unpaired samples and are indicated in the figures.FIG. 1B shows Tables 1 and 2 indicating TargetScan analysis of putativebinding site(s) of the family of four miRNAs in the 3′UTR regions ofCDK4 and CDK6, respectively. FIG. 1C (left panel) shows a scatter plotof expression of miR-149* in 11 CRC patient tumors compared to matchednormal tissue. Corresponding p-values obtained from the Wilcoxon testfor paired samples is indicated. FIG. 1C (right panel) shows a histogramof CDK4/6, p16 and Rb status in the same 11 patients. FIG. 1D shows boxplots showing the log₁₀ RNA expression of PER1 genes in the same 50normal samples as FIG. 1A compared to tumor samples. p-values wereobtained from the Wilcoxon test for unpaired samples and are indicated.

Example 3: miR-6883-5p and miR-149* Repress Expression of CDK4 and CDK6

Whether the in silico predictions regarding miR-6883-5p and miR-149*regulation of CDK4/6 translated to in vitro reults was examined A panelof CRC cell lines was used: HCT-116 (p53+/+), RKO (p53+/+), HT-29 (p53R273H), and SW-480 (p53 R273H/P309S). All the CRC cell lines used areproficient for Rb and p16 except SW-480, which is p16−/− (see FIG. 6).Each of the cell lines was reverse transfected with both miR-6883-5p andmiR-149* and examined for protein and RNA expression of CDK4 and CDK6.As shown in FIGS. 2A and 2B, miR-6883-5p targeted both CDK4 and CDK6 atboth the protein and RNA level. miR-149*, on the contrary, was morepotent in reducing levels of CDK6 and had no impact on CDK4. This datafor potency and specificity of targeting CDKs was compared with miR-206,which has previously been reported to target CDK4 (Georgantas et al.,Pigment Cell Melanoma Res., 2014, 27, 275-86). It was further confirmedusing the pMirtarget-CDK4-Luciferase vector which has the CDK4 3′UTRcloned with the luciferase gene that both miR-6883-5p and miR-149*directly bind to the 3′UTR region of CDK4. As indicated in FIG. 2C,miR-6883-5p significantly reduced luciferase signal compared to miR-206and miR-149*, respectively.

Referring in particular to FIGS. 2A, 2B, and 2C, data is presentedshowing that miR-6883-5p and miR-149* negatively regulate expression ofCD4 and CDK6 in CRC cell lines. Referring to FIG. 2A, CDK4 and CDK6protein levels were detected in a panel of CRC cell lines reversetransfected with 50 nM of miRNA mimics or scrambled duplex (SCR) for 72hours. Referring to FIG. 2B, qRT-PCR was performed for CDK4 and CDK6 inSCR or miRNA mimics transfected in CRC cell lines (50 nM, 72 hours,n=3). *, ** and *** indicate p-value relative to SCR expression.Referring to FIG. 2C, HCT-116 cells stably selected with CDK4-Luciferaseconstruct were reverse transfected with SCR or 50 nM of the indicatedmiRNA mimics for 48 hours. Measured luciferase activities werenormalized per μg of protein for indicated samples and reported asRLU±SEM (n=3). * indicates p-value relative SCR RLU.

Example 4: Restoring Expression of miR-6883-5p and miR-149* Results inG₁-Arrest and Cel Death

Given the ability to target CDK4/6, the functional consequences ofrestoring the expression of miRs-6883-5p and 149* as compared to miR-206in the panel of CRC cell lines was determined. Since the miRNAs targetproteins in the cell cycle, it was believed that they could affect cellproliferation, both short-term and long-term. As shown in FIG. 3A, allthree miRNAs had comparable and moderate short-term anti-proliferativeeffect on the panel of CRC cell lines as measured by cell viability, 72hours post-transfection. There was greater than 50-70% inhibition on thelong-term proliferation of these cells as seen from colony formationassay in all four cell lines (see, FIG. 3B). The effects of the miRNAson the cell cycle markers were also examined to determine whether theinhibition of CDK4/6 would lead to G₁-arrest in all the cell lines. Asshown in FIG. 3C, in all four cell lines, miR-6883-5p and miR-149*reduced levels of phosphorylated Rb (S795), indicating G₁-arrest andinhibition of CDK4/6 activity. These findings were further confirmed byPI-staining and looking at the cell cycle profiles of each of these celllines. As shown in FIG. 3E, both RKO and HT-29 cells showed G₁-arrest ontransfection with each of the miRNAs and a small fraction of cellsunderwent cell death.

However, in HCT-116 alone, expression of all miRNAs led to apoptosis,with between 30-50% cells in sub-G1 phase. This result furtherreplicated with the PARP-cleavage data seen in FIG. 3D. While in HCT-116cell lines, all three miRNAs induced increased apoptosis; in RKO andSW-480 cells, 6883-5p was the most potent in inducing apoptosis. Nochange in the p53 levels indicated that apoptosis was not p53-dependentin p53+/+ cells. Interestingly, miR-6883-5p downregulated XIAP andBCL_(xL), which in part could explain the induction of apoptosis bymiR-6883-5p. While XIAP is a predicted target of the family of miRNAs,BCL_(XL) is not. However, both BCL_(XL) and XIAP contribute to thepro-apoptotic effects of the miRs.

Referring in particular to FIGS. 3A, 3B, 3C, 3D, and 3E, data ispresented showing that restoring expression of miR-6883-5p and miR-149*results in G₁-arrest and cell death in CRC cell lines. Referring to FIG.3A, a panel of CRC cell lines was reverse transfected with 50 nM SCR or50 nM of indicated miRNA mimics. The effects on cell viability weremeasured 72 hours post-transfection using CellTiter-Glo assay. Referringto FIG. 3B, the effects of the miRNA mimics on long-term cellproliferation of CRC cell lines was assessed by colony formation assaysperformed in 6-well plates. Cells were reverse transfected with 100 nMof SCR or indicated miRNA mimic After 72 hours, 500 cells were seededper well in triplicate for each condition and stained with crystalviolet on Day 14. Representative images of cells stained with crystalviolet are shown (left panel) and relative colony number (n=3) isrepresented graphically (right panel). All four CRC cell lines werereverse transfected with 100 nM SCR or indicated miRNA mimics. Theeffects on cell cycle markers (see, FIG. 3C) and markers of apoptosis(see, FIG. 3D) were evaluated by western blot 72 hourspost-transfection. Representative western blots are shown (n=3).Referring to FIG. 3E, cell cycle profiles and apoptotic cells wereassessed in three CRC cell lines by reverse transfecting with SCR or 50nM (HCT-116 and HT-29) or 100 nM (RKO) miRNA mimics 72 hourspost-transfection, cells were fixed, stained with PI, and analyzed byFACS. Representative results of changes G1 and sub-G1 phases of cellcycle are graphically represented (n=3).

Example 5: Silencing of CDK4 and CDK6 Phenocopies the Effects ofmiR-6883-5p and miR-149* Mimics in CRC Cell Lines

To determine whether the biological effects of miR-6883-5p and miR-149*could be attributed to the direct targeting of CDK4/6, the expression ofCDK4/6 was silenced by siRNA and the associated functional consequenceswere detected. As shown in FIG. 4A, knockdown of CDK4 and CDK6 hadsimilar effects on short-term proliferation of CRC cell lines as withoverexpression of miRNAs. However, silencing the expression of CDK4/6was less potent in preventing long-term proliferation of CRC cell lines,especially RKO and HT-29, as seen in FIG. 4B. This indicates thattargeting of CDK4/6 by miR-6883-5p and miR-149* can only, in part,explain the anti-proliferative effects of these miRNAs. Knockdown ofCDK4 and CDK6 siRNAs arrested cells in G₁ phase of cell cycle (see,FIGS. 4C and 4D). However, unlike the miRNAs, knockdown of CDK4 alonelead to cell death HCT-116 and SW-480 cells. Given the leakiness of theCDK4 siRNA, knockdown of both CDK4 and CDK6 is more potent and needed toinduce apoptosis in CRC cell lines compared to either gene alone. Thus,the dual targeting of CDK4 and CDK6 by miRNAs has therapeutic benefitsin CRC cell lines.

Referring in particular to FIGS. 4A, 4B, 4C, and 4D, the effect ofsilencing of CDK4 and CDK6 phenocopies on miRNA mimics is shown.Referring to FIG. 4A, all four CRC cell lines were reverse transfectedwith SCR or 80 nM of CDK4 or CDK6 siRNA. The effects of short-term cellproliferation were measured 72 hours post-transfection usingCellTiter-Glo assay. Referring to FIG. 4B, the long-term effects on cellproliferation by silencing CDK4 or CDK6 were assessed by a colonyformation assay performed in 6-well plates. All four cell lines werereverse transfected with 80 nM CDK4 or CDK6. At 72 hourspost-transfection, 500 cells were seeded per well in triplicate andstained with crystal violet on Day 14. Representative images of cellsstained with crystal violet are shown (left panel) and relative colonynumber (n=3) is represented graphically (right panel). Referring to FIG.4C, all four CRC cell lines were reverse transfected with 80 nM CDK4 orCDK6 siRNA. The effects on markers of cell cycle and apoptosis wereevaluated by western blot 72 hours post-transfection. Representativewestern blots are shown (n=3). Referring to FIG. 4D, cell cycle profilesand apoptotic cells were assessed in three CRC cell lines by reversetransfecting with SCR or 40 nM (HCT-116) or 80 nM (HT-29 and RKO) siRNAof CDK4 and CDK6. 72 hours post-transfection, cells were fixed, stainedwith PI, and analyzed by FACS. Representative results of changes G1 andsub-G1 phases of cell cycle are graphically represented (n=3).

Example 6: miR-6883-5p and miR-149* Synergize with FDA-ApprovedTherapeutics for CRC

The combinatorial effect of miR-6883-5p and miR-149* with frontlinetherapeutics Irinotecan and 5-FU was evaluated. As shown in FIG. 5A,both miR-6883-5p and miR-149* synergized with Irinotecan in all fourcell lines. Further, both the miRNAs also increased the sensitivity ofp53 mutant cell lines HT-29 and SW-480 to 5-FU (see, FIG. 5B). Thesynergistic combinations led to cell death in all four cell lines asmeasured by PARP cleavage (see, FIGS. 5C and 5D). The Irinotecan-miRNAcombination engaged the intrinsic pathway of cell death as measured byCleaved Caspase-9, BCL, and XIAP (see, FIG. 5C). In cells treated with5-FU, single agent miR-149* and 5-FU caused cell cycle arrest in, asseen by p21 levels. The combination however, led to apoptosis (see FIG.5D). Thus, both miR-6883-5p and miR-149* are combination agents in CRC.

Referring in particular to FIGS. 5A, 5B, 5C, and 5D, data is presentedthat demonstrates that miR-6883-5p and miR-149* synergize withIrinotecan and 5-FU in CRC cell lines. A panel of CRC cell lines wasreverse transfected with 25 nM (HCT-116) or 50 nM of SCR or indicatedmiRNA mimics. At 16 hours post-transfection, Irinotecan (see, FIG. 5A)or 5-FU (see, FIG. 5B) at indicated doses were added. Synergy ofmiRNA-drug was measured by cell viability 72 hours post-transfectionusing CellTiter-Glo assay. The effect on apoptosis and cell cyclemarkers with miRNA alone or combination were assessed using 50 nM ofmiRNA mimic and 5 μM (HT-29 and SW-480) or 2.5 μM Irinotecan (HCT-116and RKO) by western blot. For 5-FU, 384 μM (HT-29 and SW-480) and 25 μM(HCT-116 and RKO) were used. Representative western blots are shown inFIG. 5C and FIG. 5D, respectively.

Example 7: Efficacy in Cell Lines

RNA expression data for CDK4, CDK6 and PER1 was obtained from TCGA CRCpatient samples in 50 matched tumor and normal samples (see, FIG. 8).The scatter plots indicate the log₁₀ RNA expression of normal samplescompared to tumor samples for the indicated gene of interest. p-valueswere obtained from the Wilcoxon test for paired samples and areindicated.

Cell viability, cell proliferation, and Western blot analysis ofpancreatic cancer cell lines treated with SCR or the indicated miRNA wasexamined (see, FIG. 9). Cell viability, cell proliferation, and Westernblot analysis of melanoma cancer cell lines treated with SCR or theindicated miRNA was also examined (see, FIG. 10). In addition, cellviability and Western blot analysis of the induction of G1-cell cyclearrest by targeting CDK4 and CDK6, independent of p53 status, was alsoexamined (see, FIG. 11).

Various modifications of the described subject matter, in addition tothose described herein, will be apparent to those skilled in the artfrom the foregoing description. Such modifications are also intended tofall within the scope of the appended claims. Each reference (including,but not limited to, journal articles, U.S. and non-U.S. patents, patentapplication publications, international patent application publications,gene bank accession numbers, and the like) cited in the presentapplication is incorporated herein by reference in its entirety. Thesubject matter described herein was made with government support underGrant Nos. R01 CA 176289 and P30 CA 006927 awarded by The NationalInstitutes of Health (NIH).

What is claimed is:
 1. A method for treating colorectal cancercomprising administering to a subject in need thereof one or moreoligonucleotides consisting of 15 to 40 linked nucleobases, or a saltthereof, that comprises a nucleobase sequence that is at least 80%identical to a nucleobase sequence of hsa-miR-6883-5p.
 2. The methodaccording to claim 1, wherein the one or more oligonucleotides is amodified oligonucleotide.
 3. The method according to claim 1, whereinthe one or more oligonucleotides is present in a pharmaceuticalcomposition.
 4. The method according to claim 1, wherein theoligonucleotide comprises the nucleobase sequence of SEQ ID NO:
 1. 5.The method according to claim 1, wherein the subject is also treatedwith another chemotherapeutic agent.
 6. The method according to claim 5,wherein the another chemotherapeutic agent is a platinum-basedchemotherapeutic agent, a taxane, a type I topoisomerase inhibitor, atype II topoisomerase inhibitor, or CHOP.
 7. The method according toclaim 5, wherein the another chemotherapeutic agent is Irinotecan or5-fluoruracil.
 8. The method according to claim 1, wherein the subjectis also treated with chemoembolization, radiation, and/or surgicalresection.